Overexpression, purification and characterization of a thermolabile phosphatase

ABSTRACT

Compositions for an alkaline phosphatase and methods for over-expression and purification of thermolabile Antarctic phosphatase (TAP) are provided. Uses for TAP include dephosphorylation of nucleic acids, sugars, peptides and proteins. TAP as described herein has advantages over phosphatases from other sources with respect to thermolability at 65° C. and efficiency of dephosphorylation activity at approximately neutral pH.

CROSS REFERENCE

This application is a § 371 application of international applicationnumber PCT/US04/04996 filed on Feb. 20, 2004, which claims priority fromU.S. provisional application number 60/449,711 filed on Feb. 24, 2003,herein incorporated by reference.

BACKGROUND

Purified phosphatases are used by molecular biologists to removephosphate groups from linearized DNA, RNA, nucleotides and proteins invitro. Removal of both 5′-terminal phosphate residues from a linearizedDNA vector prevents recircularization of the vector DNA in a ligationreaction because DNA ligase catalyzes the formation of a phosphodiesterbond between adjacent nucleotides only if one nucleotide contains a5′-phosphate and the other a 3′-hydroxyl group. However, foreign DNAsegments with 5′-terminal phosphates can be ligated efficiently into thedephosphorylated vector to give an open circular molecule containing twonicks that can easily be transformed into competent cells.

Several different phosphatases have been developed for removal ofphosphate groups from biological molecules, and each has its ownadvantages and disadvantages. The first phosphatase to be used for thispurpose was Bacterial Alkaline Phosphatase (BAP) purified fromEscherichia coli (E. coli). BAP has the advantage of having goodactivity against all types of DNA ends. However, it is difficult toremove from the reaction as it is very resistant to heat and detergents(Sambrook, et al., Molecular Cloning, A Laboratory Manual Sections1.53-1.72 (1989)). A mutant BAP has been prepared (U.S. Pat. No.5,891,699, EP Patent No. 0441252) which reportedly had increasedthermostability. Calf Intestinal Alkaline Phosphatase (CIP or CIAP) isanother phosphatase that has been extensively used in molecular biologytechniques (U.S. Pat. Nos. 5,773,226 and 5,707,853). CIAP is not asactive on DNA as BAP, but it is slightly easier to remove from areaction requiring the use of either Proteinase K treatment followed byphenol:chloroform extractions or a heat step in the presence of EDTAfollowed by a phenol:chloroform extraction (Sambrook, et al., supra(1989)). More recently a phosphatase isolated from Arctic shrimpPandalus borealis (SAP) (U.S. Pat. Nos. 6,387,634 and 6,379,940) hasproved easier to use. It has good activity against all types of DNA endslike BAP, but it is reported to have the advantage that it is easilyremoved from the reaction by heat inactivation at 65° C. for 15 minutes(Amersham Bioscience, Piscataway, N.J.).

Other reports have occurred in the literature for other thermolabilephosphatases including one purified from a psychrophilic strain TAB5isolated from Antarctica referred to as Thermolabile AntarcticPhosphatase (TAP) (Rina, et al., Eur. J. Biochem. 267:1230-1238 (2000)).Advantages of TAP over other phosphatases include heat lability and highspecific activity. Rina, et al. reported that this phosphatase had aspecific activity of 1650 units/mg of protein for p-nitrophenylphosphate (pNPP) substrate which was significantly higher than theactivity of any other known phosphatase. However, the protein producedby the clone reported by Rina, et al., (supra (2000)) had a number ofproblems associated with overexpression and purification. For example,the purification protocol described by Rina, et al., (supra (2000))requires multiple ultracentrifugation steps to extract the TAP from cellmembranes with which it was apparently associated. This protocol is notsuited for large scale manufacture (for example, manufacturing protocolsinvolving production of 300 g or more of cell paste). In addition,overexpressing the TAP gene using the protocol described in Rina, etal., resulted in yields of the enzyme that were very low andconsequently not cost effective for large scale manufacture.

SUMMARY

Present embodiments of the invention provide an alkaline phosphatasewith increased heat lability at neutral pH and enhanced activity and ahydrophilic leader sequence at the N-terminal end of the protein. Thehydrophilic leader sequence may be an oligopeptide, for example, anoligopeptide with a net positive charge such as a His tag. Advantages ofthis phosphatase include the ability to eliminate its activity byraising the temperature to a level at which other reactants and productsremain unaffected. An example of a phosphatase having these propertiesis TAP which is described in detail in the examples. Although the methoddescribed herein relies on the presence of a hydrophilic leadersequence, such as His tag, fused to the N-terminal end of the protein,the protein is preferably not purified using a nickel column because thenickel column buffer had an adverse effect on activity. Nonetheless, thepresence of the hydrophilic leader sequence surprisingly enhanced yieldsduring column purification using other column compositions.

In an embodiment of the invention, a truncated enzymatically active TAPis provided in which the truncation corresponds to a deletion of asignal sequence, the phosphatase having a C-terminal and N-terminal end,wherein the N-terminal end is covalently linked to a hydrophilic leadersequence, such as a positively charge oligopeptide, for example, a Histag. The truncated TAP can be substantially inactivated at about 65° C.and in less than 15 minutes. In particular, the activity of thetruncated TAP is substantially stable in Tris-HCL at a pH greater thanpH 6 more particularly at about pH 7.4.

In addition, a DNA is described that encodes TAP (also listed in GenBankY18016) but which lacks some or all of the signal sequence associatedwith the naturally occurring TAP gene. The DNA may further includesequences that encode a plurality of His amino acids preferably at theN-terminal of the expressed TAP gene. This DNA may be expressed in avector under control of a strong promoter, for example, the T7 promoter.

In a preferred embodiment, vectors are provided that contain the DNAdescribed above. An example of these vectors is pEGTAP7.4.1. Inadditional embodiments of the invention, host cells are provided whichhave been transformed with at least one vector. In the examples, thehost cell is E. coli although the use of other host cell types is notexcluded.

In additional embodiments of the invention, a formulation of TAP isprovided which is substantially stable at 37° C. in a buffer whichincludes Tris-HCL, MgCl₂, DTT and glycerol and more particularly wherethe pH is about 7.4.

In an embodiment of the invention, a method is provided foroverexpressing TAP, that includes (a) operably linking to a T7 promoter,a truncated TAP gene fused to a sequence for expressing a hydrophilicoligopeptide, such as a His tag; (b) transforming a host cell; and (c)overexpressing TAP. In further embodiments, the TAP gene encodes a TAPprotein having an N-terminal and a C-terminal end, wherein the TAPprotein is truncated at the N-terminal end, the truncation correspondingto a signal sequence, the N-terminal end having the hydrophilicoligopeptide attached hereto.

In an additional embodiment of the invention, a method is provided forobtaining a purified TAP, that includes (a) obtaining a transformed hostcell as described above; (b) disrupting the host cells to yield asoluble fraction; and (c) purifying TAP from the soluble fraction.Moreover, the method may additionally include a column separationwherein the TAP is eluted from the column. In this context, purificationrefers to an increase in the percentage of TAP protein relative to totalprotein in the disrupted cell mixture. For example, purification may beachieved when the percentage of TAP protein is more than 5% of the totalprotein in the mixture. Preferably, the percentage of TAP protein may bemore than 50%, 80% or 90% of the total protein. In a particularembodiment, the percentage of TAP protein is purified to about 95% ofthe total protein.

In an additional embodiment of the invention, a method is provided forassaying for TAP activity, that includes: (a) adding TAP to a substratein a buffer at about pH 5.5-7.0; and (b) determining the amount ofphosphate removed from the substrate in a defined time and at a definedtemperature to determine TAP activity. In a preferred embodiment, thebuffer includes ZnCl₂ and MgCl₂ salts. More particularly the buffer isBis-Tris Propane.

In an additional embodiment, a method of dephosphorylating aphosphorylated substrate is provided that includes adding TAP to thesubstrate in a buffer at pH 5.5-7.0 and causing an amount of phosphateto be removed from the substrate in a defined time and at a definedtemperature.

The substrate may be a nucleic acid or a terminal nucleotide on anucleic acid, a sugar phosphate, or a phosphorylated peptide or protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of purification of TAP from an E. coli straincontaining pN1. Lanes 1 through 6 are sequential steps in apurification. Lane 1 is total crude extract after induction. Lane 2 isthe solublized membrane fraction. Lane 3 is flow through from a DEAEcolumn. Lane 4 is pooled fractions from a Q-sepharose column. Lane 5 isflow through from a HiTrap S column. Lane 6 is flow through from aHydroxyapatite column. Lane 7 is purified TAP obtained from the pNIclone. Molecular weight standards (S) are denoted in kD. The bandcorresponding to 47.5 kD in lane 7 represents substantially purifiedTAP.

FIG. 2 shows a schematic diagram of the steps used to construct vectorsfor over-expressing the TAP gene. The rectangle at the top of the figureis a schematic drawing of the gene for TAP including an N-terminal andC-terminal portion of the corresponding expression product of the gene.At the N-terminal end, the location of the methionine start codon andthe region around the processing site encoding the hypothesized signalsequence is shown. At the C-terminal end, amino acids of the geneproduct are shown with the stop codon marked by an asterisk. Primersused in PCR amplification of the TAP gene are drawn above (not to scale)(for the forward primers A, B and C) and below (for the reverse primersD and E) the rectangle at the site at which each primer hybridizes tothe TAP gene. The N and X above the primers show engineered cleavagesites for the restriction endonucleases NdeI or XhoI. Primer C shows6H's following the NdeI site. These correspond to 6 codons encodinghistidine inserted downstream of and adjacent to the methionine startcodon in the primer to create an N-terminal His-tag fusion with TAP. Thenext set of lines following “PCR” indicate the resulting productsproduced by using the pairs of primers indicated. As above, the asteriskindicates a stop codon and the 6H's refer to 6 histidine codons. 5different plasmid are shown which were created by inserting PCR productsinto pET21a. pEGTAP1.1.1 encodes for intact TAP with no His-tag.pEGTAP1.2.1 encodes for truncated TAP with no His-tag. pEGTAP1.3.1encodes for intact TAP with a C-terminal His-tag. pEGTAP1.4.1 encodesfor truncated TAP with a C-terminal His-tag. In a later experimentpEGTAP7.4.1 was constructed to encode truncated TAP with an N-terminalHis-tag.

FIG. 3 shows an SDS-PAGE analysis of TAP produced from different clonesin which the TAP gene is either truncated or not truncated in thepresence or absence of a His-tag. “Test” purification refers to a samplesize of less than 1 ml. Lane 1 is the soluble crude extract from clonepEGTAP1.1.1 with the intact TAP gene and with no His-tag. Lane 2 is thesoluble crude extract from clone pEGTAP1.2.1 with the truncated TAP genewith no His-tag. Lanes 3 and 4 are soluble crude extracts from twodifferent isolates of the clone pEGTAP1.3.1 with the intact TAP genefused to a His-tag. Lanes 5 and 6 are soluble crude extracts from twodifferent isolates of the clone pEGTAP1.4.1 with the truncated TAP genefused to a His-tag. Molecular weight standards (S) are denoted in kD.Those clones having a truncated TAP gene (lanes 2, 5 and 6) provided thestrongest bands corresponding to TAP.

FIG. 4 shows SDS-PAGE analysis of soluble TAP purified undernon-denaturing conditions from an E. coli (ER2566) clone containing thetruncated gene with a C-terminal His-tag (pEGTAP1.4.1). 10 μl samples ofthe total crude extract (T), the clarified crude extract (load) (L), theflow-through (FT) from the Ni-NTA spin column (Qiagen, Studio City,Calif.), the wash from the Ni-NTA spin column (W) and the first andsecond samples eluted from the Ni-NTA spin column with the elutionbuffer (E1 and E2) were run on a 10-20% Tris tricine PAG (InvitrogenCorp., Carlsbad, Calif.) and stained with Coomassie Brilliant Blue.Molecular weight standards (S) denoted in kD.

FIG. 5 shows the result of a purification protocol similar to that usedin FIG. 4 except that the clone analyzed was ER2566 (pEGTAP7.4.1). Also,instead of running the total cell extract on the gel, the pelletresulting from spinning the crude extract was resuspended and run asinsoluble pellet (P). 10 μl of pellet (P), soluble load (L), flowthrough from Ni-NTA spin column (FT), wash from Ni-NTA spin column (W),elutions from Ni-NTA spin column with imidazole (E) and molecular weightstandard (S) denoted in kD were run on a 10-20% Tris tricine PAG andstained with Coomassie Brilliant Blue.

FIG. 6 shows a comparison of TAP activity at different pH and indifferent buffers. pH optimum was determined using the phosphate releaseassay. 50 mM of each buffer was incubated in a reaction mixturecontaining the ³²P labeled duplex DNA, 1 mM MgCl₂ and 0.1 mM ZnCl₂. Thereaction was incubated at 37° C. for 5 minutes and the amount oftrichloroacetic acid (TCA) soluble counts was measured.

FIG. 7 shows the effect of different salts on the activity of TAP usingthe phosphate release assay. 100 mM of each salt, KCl, NaCl, potassiumacetate or sodium acetate were tested.

FIG. 8 shows a comparison of the thermolability of CIAP, SAP and TAP.Each enzyme was incubated in the presence of DNA at 65° C. for up to 30minutes. Samples were taken at different time points during theincubation and were assayed for activity with pNPP.

FIG. 9 shows the stability of TAP at 37° C. and 25° C. by measuring therelease of phosphate from pNPP by TAP over time. Sample tubes containing1 unit of TAP and unreacted pNPP were incubated at either 25° C. or 37°C. Reactions were stopped at 5-minute intervals for 0-30 minutes byadding NaOH to a final concentration of 5 N. Release of phosphate(accumulation of pNP) was measured by spectrophotometer at 405 nm(yellow) and graphed vs reaction time.

FIG. 10 shows a comparison of the efficiency of TAP to SAP in theremoval of phosphate groups from different types of DNA termini: 5′overhang, blunt ends and 3′ overhang were tested. These ends wereproduced by digestion of the vector with HindIII, EcoRV and PstIrespectively.

FIG. 11 shows the reaction time course of deoxynucleotidase activity forTAP and SAP. The composition of the dATPase reaction mixture wasmeasured by Cap-HPLC at 5-minute intervals. The results are expressedfor each component as a percentage of the total nucleotide present.

FIG. 12 shows the release of inorganic phosphate from pyrophosphate byTAP. Increasing amounts of TAP were added to each reaction mixcontaining 0.32 mM sodium pyrophosphate. Inorganic phosphate wasmeasured essentially by the method of Heinonen and Lahti (Heinonen, J.K. and Lahti, R. J. “A new and convenient calorimetric determination ofinorganic orthophosphate and its application to the assay of inorganicpyrophosphatase.” Anal. Biochem. 113(2): 313-7, 1981) by comparison toknown phosphate standards. Thermostable inorganic pyrophosphate (TIPP)was used in a parallel reaction to demonstrate equivalence. The resultsshown are for a 10 minute assay at 37° C. for TAP and at 75° C. forTIPP.

FIG. 13 provides a table showing the specific activity of TAP ascompared to CIAP on phosphorylated myelin basic protein.

FIG. 14 shows the activity of TAP on threephospho-peptides:phospho-serine (graph A), phospho-threonine (graph B)and phospho-tyrosine (graph C) as measured by Cap-HPLC. The area of theabsorbance peaks for the phospho-peptide and the peptide are measuredand plotted on the graph as a percent of total area versus the number ofunits of TAP.

FIG. 15 shows TLC analysis of a titer of TAP on three differentsugar-phosphates, glucose-1-phosphate, glucose-6-phosphate andmannose-6-phosphate. 3 μg pf Glucose (G) and mannose (M) were spotted aspositive controls. Negative controls (−) contain just the reaction mixcontaining buffer and the sugar phosphate substrate incubated withoutenzyme. Lanes marked 1 through 6 are the serial dilution (1:1) of theenzyme in the reaction mix starting with reaction 1 containing 12.5units of TAP down to reaction 6 containing 0.4 units of TAP. Arrowsindicate the distances that the sugars and the sugar-phosphates migrateon the TLC.

FIG. 16 shows the sequence of the forward primer (SEQ ID NO:6) and thereverse primer (SEQ ID NO:8) used to PCR a 1.1 kb fragment from the pN1plasmid. The expected N-terminal amino acid sequence of the protein (SEQID NO:7) resulting from expression from the cloned PCR fragment isindicated below the sequence of the forward primer. The NdeI and XhoIcloning sites contained within the primers are also indicated.

FIG. 17 shows the DNA (SEQ ID NO:9) and amino acid sequence (SEQ IDNO:10) of the intact TAP gene. Indicated in the box is the start of theprotein as determined by N-terminal sequence analysis (Rina, et al.,supra). Arrows indicate the location at which the PCR primers used toclone the truncated gene would anneal.

DETAILED DESCRIPTION OF THE EMBODIMENTS

TAP gene has been isolated and cloned (pN1) by Rina, et al., and the DNAsequence has been published. However, when attempts were made tooverexpress and purify TAP from this clone using the published protocol,a number of problems were encountered including obtaining insufficientamounts of purified enzyme for reagent purposes and the need formultiple ultracentrifugation steps to separate the enzyme from membranefragments with which it was associated. A protocol is provided herein inwhich the TAP gene is reliably overexpressed to provide yields of enzymethat are cost effective for large scale manufacture. Furthermore, apurification protocol has been developed that avoids the need formultiple ultracentrifugation steps and is suited for large scalemanufacture. Uses for TAP have been established for removing phosphatesfrom nucleic acids, peptides, and sugars. Other substrates which may actas a substrate for TAP are those described by Reid and Wilson in “TheEnzymes” vol IV (1971) ch 17, p 373.

In a preferred embodiment, “sugars” here refer to monosaccharides,oligosaccharides or polysaccharides. Sugars include any pentose, hexoseor heptulose compound. A phosphate may be cleaved using TAP from anycarbon in the sugar wherever the phosphate or phosphates are positionedirrespective of whether the phosphate is an alpha or beta isomer.Examples of some sugar phosphates are provided at the web sitehttp://www.arabidopsis.org. Examples of sugar phosphates include:phosphates of mannose, glucose, fucose, galactose,N-acetylgalactosamine, N-acetylgluosamine, xylose, and rhamnose.

Purification of TAP from ER2575 (ER2566 pLysS) containing the pN1plasmid was attempted using modifications of the published protocol toimprove yield and ease of purification. The transformed E. coli strainwas grown and induced and the phosphatase was purified as described inRina, et al. (supra), with the following exceptions. After the solublemembrane fraction was isolated by ultracentrifugation, the proteins weredialyzed in buffer A (20 mM Tris pH 7.6, 10 mM MgCl₂, 50 mM NaCl, 0.2%Triton X-100) then loaded onto a DEAE column which had been equilibratedwith buffer A. The flow-through which had the phosphatase activity asdetermined with a pNPP assay was loaded on to a Q-sepharose column asdescribed in Rina, et al. The pooled fractions from the Q-sepharosecolumn were dialyzed in buffer B (Potassium phosphate buffer pH 6.6, 25mM NaCl, 10 mM MgCl₂) and loaded on a Hi-trap SP column which had beenequilibrated in buffer B. The flow-through which had the phosphataseactivity was dialyzed in buffer C (Potassium phosphate buffer pH 7.2, 50mM NaCl, 10 mM MgCl₂) and loaded onto a hydroxyapatite column which hadbeen equilibrated with buffer C. The phosphatase activity flowedthrough. Samples from each step were run on a 10-20% Tris tricine PAG(Invitrogen Corp., Carlsbad, Calif.) and stained with CoomassieBrilliant Blue. As can be seen in FIG. 1, the yield of phosphatase inlanes 1-6 was disappointing.

With the disappointing results obtained using the above protocols,alternative approaches were investigated that involved cloning the TAPgene as described in FIG. 2.

Specifically, the TAP gene was cloned directly behind a strong promoter(T7 promoter) and attached a His-tag (affinity tag) to facilitatepurification. The His-tag sequence shown in FIGS. 13 and 14 has sixhistidines fused to the N-terminal end of the protein (5′ end of thegene). However, this number of histidines is not critical. The number ofhistidines in the tag may be as few as three or as many as desired inexcess of six to achieve the purpose described herein. Several differentconstructs were made. In two constructs, the TAP gene was truncated byremoving a putative signal sequence consisting of 22 amino acids at theN-terminal end of the protein adjacent to the methionine start codon. AC-terminal His-tag was added to form a fusion protein with the truncatedphosphatase for one clone but not for another. For example, pEGTAP1.4.1had a His-tag while pEGTAP1.2.1 did not. In two other constructs, thegene retained the putative signal sequence and either additionallycontained a C-terminal His-tag (pEGTAP1.3.1) or not (pEGTAP1.1.1) (FIG.2).

Five different plasmids are shown in FIG. 2 which were created byinserting PCR products into pET21a (Example I, FIGS. 2, 11 and 12).pEGTAP1.1.1 encodes for intact TAP with no His-tag. pEGTAP1.2.1 encodesfor truncated TAP with no His-tag. pEGTAP1.3.1 encodes for intact TAPwith a C-terminal His-tag. pEGTAP1.4.1 encodes for truncated TAP with aC-terminal His-tag. pEGTAP7.4.1 encodes truncated TAP with an N-terminalHis-tag.

Plasmids containing the intact TAP gene with the putative signalsequence, with or without the His-tag at the C-terminal end, failed toproduce any obvious soluble phosphatase on SDS-PAGE after induction(FIG. 3, lanes 1, 3 and 4). However, those plasmids constructed withoutthe putative signal sequence either with or without the His-tag produceda very strong band of the correct size on SDS-PAGE (FIG. 3, lanes 2, 5and 6). However, problems occurred during purification of the protein.The transformed host cell ER2566 containing pEGTAP1.4.1 (truncated TAPand C-terminal His) produced predominantly insoluble TAP innon-denaturing conditions (FIG. 4). Purification was attempted underdenaturing conditions followed by refolding of the denaturedphosphatase. However, this approach yielded relatively small amounts ofactive enzyme. E. coli ER2566 containing pEGTAP1.2.1 (truncated TAP withno His-tag) produced soluble TAP, however, the protein failed to bind tomost columns.

To overcome the above problems, a fifth clone was constructed in which asequence encoding His-tag was placed at the N-terminal end instead ofthe C-terminal end of the TAP gene (pEGTAP7.4.1) (FIG. 2). Thisconstruct produced good yields of TAP in the soluble fraction (FIG. 5)which could be readily purified without multiple ultracentrifugationsteps (Example II). Specifically, the presence of His-tag at theN-terminal end of the phosphatase enabled purification to nearhomogeneity on only 2 or 3 columns not including a nickel column. Whilenot wishing to be limited by theory, it appears that the primary use ofthe His-tag was to facilitate production of soluble phosphatase ratherthan as an affinity tag.

The activity of the TAP phosphatase prepared by the above method(Example I and II) was compared with SAP and CIAP phosphatase activityby means of assays employing DNA and protein substrates. (Example VII)and TAP was found to have enhanced activity. For example, linear DNAdephosphorylation activity of TAP and SAP was determined using aphosphate release assay described in Example VII, for 5′ overhangs', 3′overhangs and blunt ends of DNA. When the dephosphorylation of thesedifferent structures were compared using identical pNPP unit amounts,TAP proved to be consistently more efficient than SAP (FIG. 10).

TAP is also able to remove phosphate groups from serine/threonine andtyrosine residues on phosphorylated proteins as efficiently as CIAP(Example VII, FIG. 13).

In addition to increased activity of TAP compared with otherphosphatases, TAP was found to have enhanced thermolability (Example IV,FIG. 8). When the heat lability of CIAP, SAP and TAP was compared, itwas found that TAP had lost 98% of its activity after 5 minutes at 65°C. compared with SAP which had lost 70% activity and CIAP which had lostonly 10% of its activity at this temperature.

In other embodiments of the invention, ZnCl₂ and MgCl₂ were found toenhance the activity of the phosphatase (Example III). The activity ofTAP was optimized at a pH of about 5.5-7.0, for example, using Bis-TrisPropane buffer (Example III).

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

The references cited above and below are hereby incorporated byreference herein.

EXAMPLES Example I Construction and Expression of a TAP His-tag FusionGene

The truncated phosphatase gene derived from an unclassified Antarcticstrain TAB5 was cloned with an N-terminal 6-histidine residue tag.Briefly, a 1.1 kb fragment was amplified by PCR from the recombinantplasmid pN1 (Rina, et al., Eur. J. Biochem. 267:1230-1238 (2000)). Theforward primer (FIG. 2 primer C), in addition to containing the sequencehomologous to the 5′ end of the gene without the putative signalsequence (63 bp) (FIGS. 16 and 17), contained the NdeI restriction siteas part of the ATG start and 6 codons encoding for 6 histidine residuesplaced between the ATG start and the first codon of the truncatedphosphatase gene (FIGS. 16 and 17). The reverse primer (FIG. 2 primerD), in addition to containing the sequence reverse complementary to the3′ end of the gene, contained an XhoI restriction site immediatelydownstream of the TAA stop codon (FIGS. 16 and 17). Once amplified, the1.1 kb fragment was digested with NdeI and XhoI and ligated into the T7expression vector pET21a (Novagen, Madison, Wis.) similarly digestedwith NdeI and XhoI (Sambrook, et al., Molecular Cloning, A LaboratoryManual, Sections 1.53-1.72 (1989)). The resulting ligation wastransformed into the E. coli strain ER2688 (New England Biolabs,Beverly, Mass.). Plasmids from the transformation were isolated andcharacterized by restriction mapping and DNA sequencing and whencharacterized proved to contain an insert of the correct size andsequence. The plasmid constructs were transformed into E. coli strainER2566 (New England Biolabs, Beverly, Mass.) for expression of thephosphatase gene from the T7 promoter. pEGTAP7.4.1 was found to providethe best yield and purification profile (FIGS. 3-5).

Example II Production and Purification of the TAP His-tag Fusion Protein

(a) Small Scale Purification of TAP

ER2566 (pEGTAP7.4.1) was grown overnight at 30° C. from an isolatedcolony in 5 ml Rich Broth ((10 g Tryptone (Difco Laboratories, Livonia,Mich.), 5 g Yeast Extract (Difco Laboratories, Livonia, Mich.), 5 g NaClph to 7.2 with NaOH per liter)) with 100 mg/ml carbenicillin. 3 ml ofthe overnight culture was used to inoculate 300 ml of Rich Broth with100 mg/ml carbenicillin. The culture was grown at 30° C. to mid log(Klett 70) and chilled on ice, induced with 0.4 mMisopropyl-β-D-thiogalactoside (IPTG) and grown at 15° C. for 20 hours.35 ml of cells were harvested by centrifugation at 8000 rpm for 10minutes at 4° C. The supernatant was removed, the cell pellet wasweighed and placed at −20° C. for 2 hours. The pellet was thawed on iceand the cells were resuspended in 1 ml lysis buffer (50 mM NaH₂PO₄, 300mM NaCl, 10 mM imidazole pH 8.0). Lysozyme was added to 1 mg/ml and thesample was incubated on ice for 30 minutes. Following incubation thecells were broken by sonication on ice for 6 times at 10 seconds eachtime with a 1 minute rest half way through. The crude extract was spunat 14,000 rpm for 20 minutes at 4° C. to remove any unbroken cells andany other insoluble material to generate a clarified crude extract. 0.6ml of the clarified crude extract was loaded on to a Ni-NTA spin columnwhich had been equilibrated with 0.6 ml lysis buffer. The sample wascentrifuged at 700×g for 2 min. The flow-through was removed and thecolumn was washed with 2×0.6 ml wash buffer (50 mM NaH₂PO₄, 300 mM NaCl,20 mM imidazole pH 8.0). The protein was eluted with 2×0.2 ml elutionbuffer (50 mM NaH₂PO₄, 30 mM NaCl, 250 mM imidazole pH 8.0). 10 μl ofeach step were analyzed on SDS-PAGE. The results for pEGTAP7.4.1 areshown in FIG. 5.

(b) Large Scale Purification of TAP from pEGTAP7.4.1 in ER2566

E. coli strain ER2566 harboring the pEGTAP7.4.1 plasmid was grown at 25°C. in 2000 ml Fermentation Rich Broth (500 g Amberex yeast extract(Sensient Technologies, Milwaukee, Wis.), 1 kg CE90MS Tryptone (MarcorDevelopment Corp., Carlstadt, N.J.), 27 g NaOH, 10 g ampicillin, 5.5 gpolypropylene glycol antifoam agent (Arco Chemical Co., SouthCharleston, W. Va.) per 100 liters) at 25° C. This culture was used toinoculate 100 liters of Fermentation Rich Broth. The cells were grownaerobically at 25° C. for 5 hours until they reached a Klett of 70. Thefermentor was then cooled to 15° C. and IPTG was added to a finalconcentration of 0.3 mM when the Klett reached 90. The cells were thengrown aerobically at 15° C. for a further 17 hours, reaching stationaryphase with a final Klett of 305. One 100 liter fermentation was requiredto harvest 321 grams of wet cell pellet. The 321 gram cell pellet wassuspended in 963 ml buffer A (20 mM Potassium phosphate buffer (pH 7.4),50 mM NaCl, 5% Gycerol) and passed through a Gaulin homogenizer at˜12,000 psig. The lysate was centrifuged at ˜13,000×G for 40 minutes andthe supernatant collected (1150 ml).

The supernatant solution was applied to a 500 ml Heparin Hyper-D column(Ciphergen Biosystems, Inc., Fremont, Calif.) equilibrated in buffer A.A 1.0 L wash of buffer A was applied, then a 2 L gradient of NaCl from0.05 M to 1 M in buffer A was applied and fractions of 50 ml werecollected. Fractions were assayed for phosphatase activity by incubatingsamples with 0.1 M pNPP in 1M diethanolamine/HCL buffer (pH 8.5)containing 10 mM MgCl₂. Reactions were carried out at 37° C. for 1-5minutes and activity was measured as generation of yellow colorspectrophotometrically at 405 nm. Phosphatase activity eluted from0.05-0.35 M NaCl.

The Heparin Hyper-D column fractions containing the phosphatase activitywere pooled, then dialyzed against buffer A overnight. 100 ml of this800 ml pool was applied to a 105 ml Source Q column (Pfizer, Inc., N.Y.,N.Y.). A 210 ml wash with buffer A was applied followed by a 1.0 Lgradient from 0.05 M to 1.0 M NaCl in buffer A and fractions of 15 mlwere collected. Fractions were assayed using the pNPP assay describedabove and the phosphatase activity eluted from 0.15-0.18 M NaCl. Theremaining 700 ml of the Heparin Hyper-D pool was similarly applied andeluted; then the gradient fractions containing phosphatase activity werepooled.

The combined Source Q pool was dialyzed against buffer A andsupplemented with 50% glycerol. Forty ml of this 120 ml pool was dilutedto 500 ml with buffer A, then applied to a 400 ml PEI column (Whatman,Kent, U.K.) which had been pre-equilibrated with buffer A. A wash of 400ml buffer A was applied followed by a linear gradient from 0.05 M to 1.0M NaCl in buffer A and fractions of 15 ml were collected. Fractions wereassayed using the pNPP assay described above. Phosphatase activity waseluted between 0.14 M and 0.2 M NaCl. The remaining 80 ml of the SourceQ pool was similarly applied and eluted; then the gradient fractionscontaining phosphatase activity were pooled. This pool was dialyzed intostorage buffer containing 10 mM Tris (pH 7.4), 1 mM MgCl₂, 1 mM DTT, 50%glycerol.

Example III Determination of Optimal pH and Salt Conditions for Removalof a Phosphate Group by TAP

Using TAP purified as described in Example II, the optimum conditionsfor removal of a phosphate group from DNA was determined.Dephosphorylation was measured by means of a phosphate release assay asfollows: In a 50 μl reaction, two complementary 40mer oligonucleotides

-   5′-ACGTATGTTAGGTTAGGTTAGGTTAGGTTAGGTTAGGCTC-3′ (SEQ ID NO:1)-   3′-TGCATACAATCCMTCCMTCCMTCCMTCCAATCCGAG-5′ (SEQ ID NO:2)    were annealed and end-labeled as recommended by the manufacturer    using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly,    Mass.) and gamma ³²P-ATP. This radioactive dimer was used to ‘spike’    1 mg of a mixture of lambda HindIII fragments which served as a    phosphatase substrate (20 μg/ml final). TAP activity was determined    by release of radioactivity following incubation of the phosphatase    with the substrate mix (0.01 pNPP units per reaction in a buffer    containing 1 mM MgCl₂ and 0.1 mM ZnCl₂) at 37° C. for 5 minutes.    Release was measured by precipitation of the DNA substrate with TCA    and scintillation counting the radioactivity which was not    precipitable. TCA precipitation consisted of adding 100 μl of    Herring Sperm DNA (2 mg/ml) to each reaction as carrier. 150 μl of    20% cold TCA was then added. The samples were vortexed and chilled    on ice for 5 minutes. Each reaction was centrifuged in a microfuge    for 5 minutes at 14,000×g. 150 μL of each supernatant (50%) was    added to 2 ml of scintillant and counted for 0.5 minutes. TAP    activity was found to be linear over the range of 0.001 to 0.01    units measured by the pNPP calorimetric assay.

A reaction buffer with a pH range of 5.5 to 9.5 was tested using threedifferent buffers: Tris-HCL pH range 6 to 9.5, Bis-Tris Propane bufferpH range 6 to 7 and Bis-Tris Propane buffer pH range 5.5 to 7. The pHrange which gave optimum activity on a DNA substrate in Bis-Tris Propanebuffer proved to be between 5.5 and 6.0 (FIG. 6).

The cation requirement was also tested. It was determined that contraryto Rina, et al., (supra (2000)) which reported that Zn²⁺ ions wereinhibitory in assays on pNPP, the assay described above revealed thatthe presence of ZnCl₂ gave a ten-fold increase phosphate release. 1 mMMgCl₂, 0.1 mM ZnCl₂ was subsequently included in TAP activity assays.Addition of other salts including KCl, NaCl, potassium acetate andsodium acetate at concentrations of 100 mM were observed to have noeffect on TAP activity (FIG. 7).

Example IV Determination of Thermolability of TAP

A unit of pNPP activity corresponds to the amount of enzyme required tohydrolyze 1 μmol of pNPP to p-nitrophenol in a reaction volume of 1 mlin 1 minute at room temperature. Ten pNPP units each of TAP, CIAP andSAP phosphatase were mixed with 1 μg lambda HindIII fragments in therecommended reaction buffer for each enzyme. The mixture was incubatedat 37° C. for 10 minutes and then placed on ice. The reactions were thenplaced in a 65° C. water bath with samples removed and placed on iceafter 5, 10, 20 and 30 minutes. Following heat treatment the sampleswere assayed for pNPP activity. Activity remaining was calculated as apercentage of the 0 time point. After 5 minutes the TAP had lost greaterthan 98% of its activity. In that same time period SAP still had 30%activity remaining and CIAP had almost 90% activity remaining. After 30minutes SAP still had 20% activity remaining and CIP had 40% remainingshowing that TAP is much more heat labile than either SAP or CIAP (FIG.8).

Example V Stability of TAP at 37° C. and 25° C.

The stability of TAP activity at 37° C. and 25° C. was measured byrelease of free phosphate from pNPP in a colorimetric assay. Activity inthis assay was determined by the generation of color that occurs whenpNPP is converted to p-nitrophenol (pNP). A phosphatase reaction mix wasprepared containing 1 M diethanolamine, (pH 8.5), 10 mM MgCl₂, 10 mMpNPP and 10 units TAP per ml. This mix was aliquoted to 3 ml reactiontubes, 0.1 ml per tube, and the reactions were initiated by placing thetubes at 25° C. or 37° C. Reactions were terminated at 5-minuteintervals from 0-30 minutes by adding 0.1 ml of 10 N NaOH. Each reactionwas diluted with 1 ml of reaction mix (without pNPP or TAP) to bring itto a suitable volume for spectrophotometry at 405 nm. Released phosphatewas determined by measuring accumulated pNP and comparison to a knownpNP standard. The results were expressed as nmoles phosphate released ateither 25° C. or 37° C. over a 30 minute time course (FIG. 9). Theseresults demonstrated that TAP activity is nearly two-fold higher at 37°C. than at 25° C. and that TAP is stable for at least 30 minutes ateither temperature.

Example VI Optimal Conditions for Storage of TAP

TAP was incubated at 75° C. for 5 minutes to determine optimumconditions for storage. Three different buffers were tested, Bis-TrisPropane buffer pH 6.0, Phosphate buffer pH 7.0 and Tris-HCL buffer pH7.4, the latter proved the best at stabilizing the TAP. Differentconcentrations of NaCl were tested with no NaCl proving better than 50,100 or 200 mM at maintaining enzyme activity. Where EDTA was inhibitoryto TAP enzyme activity, both 1 mM DTT and 200 μg/ml BSA stabilized TAPactivity. In a buffer containing Tris-HCL (pH 7.4), 1 mM MgCl₂, 1 mMDTT, 200 μg/ml BSA and 50% glycerol TAP was found to be stable for over12 months with no decline in activity.

Example VII Phosphatase Activity

(a) Comparison of the Enzyme Activity of TAP to SAP in a DNA LigationAssay Normalized for pNPP Activity:

Dephosphorylation of 5′, 3′ and blunt ends.

Efficiency is defined as the amount of DNA which can be dephosphorylatedby 1 pNPP unit of enzyme based on the dilution which can dephosphorylate1 μg of DNA.

SAP and TAP were both adjusted to 1 unit per μl using the pNPP assay.Litmus 28 DNA (New England Biolabs, Inc., Beverly, Mass.) was digestedwith either HindIII (5′ overhang), EcoRV (blunt) or PstI (3′ overhang).Aliquots of each cut vector DNA (1 mg/50 μl) were treated with severaldilutions of each phosphatase for 30 minutes at 37° C. in theirrecommended buffers. TAP was heat killed for 5 minutes at 65° C. SAP washeat killed for 15 minutes at 65° C. Cut and dephosphorylated DNAs werethen recircularized using the Quick Ligase Kit (New England Biolabs,Inc., Beverly, Mass.) according to the instructions. Ligated vectorswere then transformed into E. coli and plated on ampicillin platesovernight. Phosphatase activity was considered complete if the number ofcolonies was less than 5% of the control (vector cut but notdephosphorylated). Whereas 1 pNPP unit of SAP could dephosphorylate 5 μgof 5′ overhang DNA, the same number of pNPP units of TAP coulddephosphorylate 50 μg of the same DNA (FIG. 10). TAP was therefore shownto be 10 times more efficient at removing phosphate groups from 5′overhangs on DNA. On blunt ends TAP was 50 times more efficient than SAPand on 3′ overhangs TAP was 8 times more efficient.

(b) TAP can Remove Phosphate Groups from Deoxynucleotides.

The activity of TAP as a deoxynucleotidase was measured by release offree phosphate in a colorimetric assay essentially as described byHeinonen and Lahti (supra). Activity in this assay was determinedrelative to known phosphate standards. 0.1 ml of four differentdeoxynucleotide reaction mixes containing 50 mM Bis Tris-Propane (pH6.0), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 1 mM Sodium dATP, dCTP, dGTP or TTPwere placed into four separate 1.5 ml tubes. 2.5 units of TAP were addedto each tube. The reactions were initiated by placing the tubes at 37°C. and terminated after 15 minutes by placing the tubes at 65° C. for 5minutes. The entire contents of each reaction mix tube (0.1 ml) wasadded to a glass tube containing 2.4 ml of measurement solutioncontaining 1.25 N H₂SO₄, 4 mM Ammonium Molybdate and 50% acetone. Thetube was vortexed briefly and incubated at room temperature for 15minutes. The measurement reactions were stopped by adding citric acid toa final concentration of 0.33 M and the OD₃₉₀ of each tube was recorded.A standard curve was generated by adding 10 μl each of 0-30 mM phosphatestandards to mock reaction tubes which were treated and measuredidentically to TAP reaction tubes. It was determined that dATP, dCTP,dGTP and TTP were nearly equivalent substrates for TAP; generating 1-2nmoles of phosphate per minute per unit under these conditions.

The deoxynucleotidase activities of SAP and TAP were compared. dATP wasused as the test substrate, but it was concluded that similar resultswould be obtained using dCTP, dGTP or TTP. A deoxynucleotidase reactionmix was prepared containing 50 mM Bis Tris-Propane (pH 6.0), 1 mM MgCl₂,0.1 mM ZnCl₂ and 0.8 mM dATP. 0.1 ml of this mix was aliquoted intoseven 1.5 ml reaction tubes. 5 units of either TAP or SAP were added toeach tube. The reaction was initiated by placing the tubes at 37° C. andterminated at 5-minute intervals for 0-30 minutes by placing the tubesat 65° C. for 5 minutes. Reaction products were subjected to capillaryhigh-performance liquid chromatograph (Cap-HPLC) for a comparison to aprofile containing standards for dATP, dADP, dAMP and deoxyadenosine. AnAgilent 1100 Series Cap-HPLC equipped with an automated sample injector,column heater and a diode array detector was used to perform allanalysis. Standards of dATP, dADP, dAMP and dA were purchased from SigmaChemicals. A 3 μm, 150×1 mm C18 reverse-phase Cap-HPLC Develosil® column(Nomura Chemical Co. Ltd., Aichi, Japan) was used to separate the fourspecies. An isocratic separation using 95% 0.1 M K₂HPO₄, pH 6.0 with KOHand 5% acetonitrile at a flow rate of 20 μl/min at 30° C. using a 100 μlflow sensor was found to produce a good resolution of all four species.Typical retention times for the dATP, dADP, dAMP and dA were 4.7, 5.2,7.0 and 21.6 minutes, respectively. TAP-and SAP-treated samples werediluted to 0.2 ml in the above buffer and 4 μl were injected per run.Data was collected using Agilent ChemStation Software (AgilentTechnologies, Palo Alto, Calif.) for the 254 nm and 280 nm absorbanceand the peak sizes were quantitated by the software. The results wereexpressed for each reaction component as a percentage of the totalnucleotide present (FIG. 11). These results demonstrated that TAP wascapable of functioning as a deoxynucleotidase, removing phosphate groupsfrom dNTP, dNDP or dNMP (where N represents adenosine, cytosine,guanosine or thymidine) and that TAP had a higher specific activity as adeoxynucleotidase than SAP (Heinonen and Lahti (supra)).

(c) TAP can Release Inorganic Phosphate from Pyrophosphate.

The activity of TAP as a pyrophosphatase was measured by release of freephosphate in a colorimetric assay essentially as described by Heinonenand Lahti (supra). Activity in this assay was determined relative toknown phosphate standards using a known pyrophosphatase as a control. Apyrophosphatase reaction mix was prepared containing 50 mM BisTris-Propane (pH 6.0), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 0.32 mM Sodiumpyrophosphate. This mix was aliquoted to 1.5 ml reaction tubes, 0.5 mlper tube and serial 2-fold TAP dilution was performed, starting with 10units in the first tube. The reaction was intitiated by placing thetubes at 37° C. and terminated after 10 minutes by placing the tubes at65° C. for 5 minutes. The phosphate measurement solution was preparedcontaining 1.25 N H₂SO₄, 4 mM Ammonium Molybdate and 50% acetone. Theentire contents of each reaction mix tube (0.5 ml) was added to a glasstube containing 2 ml of measurement solution, vortexed briefly andincubated at room temperature for 15 minutes. The measurement reactionswere stopped by adding citric acid to a final concentration of 0.33 Mand the OD₃₉₀ of each tube was recorded. A standard curve was generatedby adding 10 μl each of 0-30 mM phosphate standards to mock reactiontubes which were treated and measured identically to TAP reaction tubes.A reaction containing TIPP was run in parallel. The TIPP reaction mixcontained 2 units of TIPP, 50 mM Tricine (pH 8.0), 1 mM MgCl₂ and 0.32mM Sodium pyrophosphate; and was incubated at 75° C. This reaction wasstopped after 10 minutes by placing the tube at room temperature whereTIPP is inactive. Measurement of released phosphate was performed aswith TAP. This experiment showed that 10 units of TAP releaseapproximately the same amount of inorganic phosphate as 2 units of TIPP(FIG. 12). These results demonstrate that TAP is capable of functioningas a pyrophosphatase, cleaving pyrophosphate (PPi) into inorganicphosphate (Pi).

(d) TAP Removes Phosphate Groups from Phosphorylated Peptides.

Preparation of ³³P labeled Myelin Basic Protein substrate (MyBP).

Serine/threonine (Ser/Thr) phosphorylated MyBP substrate was preparedusing the Protein Serine/Threonine phosphatase (PSP) Assay System kitfrom New England Biolabs, Inc., Beverly, Mass. (catalog number #P0780S).Ser/Thr labeled MyBP substrate was diluted to a concentration of 50 μM(5× concentrated) with respect to the incorporated ³³P.

Tyrosine (Tyr) phosphorylated MyBP substrate was prepared using theProtein Tyrosine phosphatase (PTP) Assay System kit from New EnglandBiolabs, Inc., Beverly, Mass. (catalog number #P0785S). Tyr labeled MyBPsubstrate was diluted to a concentration of 25 μM (5× concentrated) withrespect to the incorporated ³³P.

10 μl of either Ser/Thr (50 μM) or Tyr labeled MyBP protein substrate(25 μM) was incubated with serial dilutions of the TAP phosphatase, 10pNPP units of CIAP phosphatase or no enzyme in the recommended bufferfor 1 hour at 37° C. The reactions were terminated by adding 200 μl ofcold 20% TCA and incubating on ice for 5-10 minutes. The samples werethen centrifuged at 12,000×g for 5 minutes. 150 μl of the supernatant iscarefully removed and added to 2 mis of scintillation fluid and countedin a scintillation counter. The results are shown in FIG. 13.

The specific activity for TAP against phosphorylated Ser/Thr residues is1910 nmol/min/mg which is slightly higher than the specific activity forCIAP against the same substrate. The specific activity for TAP againstphosphorylated Tyr residues is also greater than the specific activityfor CIAP (FIG. 13).

The results of the above experiment the activity of TAP were confirmedusing three peptide substrates that each contained a singlephospho-amino acid residue. These peptides were prepared using standardFMOC-chemistry. Peptide 1 contained a phosphoserine residue, Peptide 2contained a phosphothreonine residue and Peptide 3 contained aphosphotyrosine residue indicated by pSer, pThr and pTyr, respectively.Listed below are the sequences for each peptide.

1. H-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp- (SEQ ID NO:3)Arg-Arg-Val-pSer-Val-Ala-Ala-Glu- NH₂ 2. H-Thr-Ala-Asp-Ser-Gln-His-Ser-(SEQ ID NO:4) pThr-Pro-Pro-Lys-Lys-Lys-Arg-Lys- Val-Glu-OH 3.H-Glu-Trp-Met-Arg-Glu-Asn-Ala-Glu- (SEQ ID NO:5) pTyr-Leu-Arg-Val-Ala-OH

Control peptides were also synthesized which had identical sequences butlacked the phosphate in each peptide.

A 50 ul reaction containing 5 ug of each phospho-peptide in 1×TAP buffer(see Example VIII) was incubated at 37° C. for 10 min with a serialdilution of TAP. After incubation Cap-HPLC was used to analyzed thesamples. All three peptide de-phosphorylation reactions were analyzedusing a Vydac Sum, C18 1×250 mm column at a flow rate of 25 μl/min.However an optimized gradient was required to separate thephosphorylated and dephosphorylated peptides for each peptide set. TheCap-HPLC gradient program timetables using an increasing percentage ofacetonitrile (% B) relative to 0.1% TFA in water (v/v) were as follows:

TABLE 1 Time (mins) % B Peptide 1  0-10 gradient from 20%-35% 10-30gradient from 35-95% Time % B Peptide 2  0-10 gradient from 8.5%-35%10-30 gradient from 35%-95% Peptide 3  0-10 gradient from 5%-25% 10-30gradient from 25%-55% 30-60 gradient from 55%-95%

A ratio of 80% A and 20% B, 91.5% A and 8.5% B and 95% A and 5% B wasused to equilibrate the column for peptides 1, 2 and 3 respectively.Data was collected using Agilent ChemStation Software (AgilentTechnologies, Palo Alto, Calif.) for the 214 nm, 254 nm and 280 nmabsorbances and the peak sizes were calculated by the software. Toverify the identity of the species present in the reactions thereactions were also examined on Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS).An ABI Voyager DE Mass Spectrometer was used withα-cyano-4-hydroxycinnamic acid as the matrix, an accelerating voltage of20 kV and a delay time of approximately 150 ηsec in positive ion mode.FIG. 14 consists of three graphs of the compiled data showing thedecreased amount of the phospho-peptide and increasing amount ofdephosphorylated peptide with increasing amounts of TAP. This data showsthat TAP is most active on the phospho-tyrosine peptide with only 0.02units of enzyme being required to remove all the phosphates from 5 μg ofthe peptide in 10 mins. 2.5 units and 10 units of TAP were required tocompletely remove the phosphates from the phospho-serine andphospho-threonine peptides respectively. This data shows that TAP can beused to remove phosphate groups from peptides.

Example VIII TAP can Dephosphorylate Sugar Phosphates Regardless of thePosition of the Phosphate on the Sugar

To test if TAP could cleave the phosphate residue from a sugar phosphateirrespective of position, its activity for three sugar phosphatesglucose-1-phosphate, glucose-6-phosphate and mannose-6-phosphate wasdetermined. A dilution series of the reaction mixture was prepared foreach sugar phosphate where each reaction mixture contained 100 μl of the5 mg/ml solution of one of the sugar phosphates, 30 μl of the 10×TAPBuffer (500 mM Bis Tris-Propane, 10 mM MgCl₂, 1 mM ZnCl₂, pH 6.0) and210 μl H₂O. 50 μl of the reaction mix was added to the first tube and 25μl of the reaction mix to five subsequent tubes. This was repeated foreach of the three sugar phosphates to be tested. These tubes were placedon ice and allowed to chill. Once cold 5 μl of the TAP (5 U/μl) wasadded to the first tube containing 50 μl reaction mix. After mixing 25μl was removed from this tube and placed in the next tube whichcontained 25 μL of the reaction mix. After mixing 25 μl was removed fromthis second tube and placed in the next tube. This 1:1 dilution serieswas continued for a total of 6 tubes. After a dilution series of the TAPhad been prepared for each sugar phosphate, the tubes were removed fromthe ice and placed at 37° C. for 15 minutes. After incubation 3 μl ofeach sample was spotted in a tight band on silica gel glass backed thinlayer chromatography (TLC) plate. Negative controls containing just themix incubated with no enzyme and positive controls containing 1 mg/mlglucose or mannose were also spotted on the TLC. The spots werecompletely dried with a hot air gun (temperature not exceeding 70° C.).The plate was developed until the solvent front moved 9 cm inisopropanol, ethanol and water (2.5:1:0.5 v:v:v). Followingchromatography, the plate was dried and sprayed with 10% perchloricacid. The sugars were visualized by charring the plate with a heat gun.Increased sensitivity was achieved by visualizing the products under UVat 366 nm. Complete digestion of 42 μg of glucose-1-phosphate,glucose-6-phosphate and mannose-6-phosphate was achieved in 15 minutesat 37° C. using 6 U of TAP (FIG. 15).

The linkage of the phosphate on the sugar (i.e. 1 or 6) had no apparenteffect on the activity of the enzyme. In addition the activity of theTAP on mannose sugar phosphates was the same as on glucose sugarphosphates.

1. A method of dephosphorylating a phosphorylated substrate, comprising:(a) adding the thermolabile Antarctic phosphatase (TAP) of SEQ ID NO: 10to the substrate in a buffer at pH 5.5-6.5; and (b) incubating themixture comprising the substrate and the TAP under conditions to allowphosphate to be removed from the substrate.